Electrophoresis is a technique used to separate charged species on the basis of size, electric charge, and other physical properties. In electrophoresis, the charged species migrate through a conductive electrophoretic medium, which may be (but is not required to be) a gel, under the influence of an electric field. Activated electrodes located at either end of the electrophoretic medium provide the driving force for the migration. The properties of the molecules, including their charge and mass, determine how rapidly the electric field causes them to migrate through the electrophoretic medium.
Many important biological molecules, such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionizable groups. Because of these ionizable groups, at any given pH, many important biological molecules exist in solution as electrically charged species. The electrically charged species enable doctors and scientists to separate nucleic acids and proteins using electrophoresis.
Separation of molecules, biological or otherwise, using electrophoresis depends on various forces, including charge and mass. When a biological sample, such as a protein or DNA, is mixed in a buffer solution and applied to an electrophoretic medium these two forces act together. Separation using electrophoresis is possible because the rate of molecular migration through the electric field depends on the strength of the field, the size and shape of the molecules, and the ionic strength and temperature of the buffer through which the molecules are moving. During electrophoresis, the applied electrical field causes the molecules to move through the pores of the electrophoretic medium based on the molecular charge. The electrical potential at one electrode repels the molecules while the potential at the other electrode simultaneously attracts the molecules. The frictional force of the electrophoretic medium also aids in separating the molecules by size. After the applied electrical field has been removed, the molecules may be stained. After staining, the separated macromolecules can be seen in a series of bands spread from one end of the electrophoretic medium to the other. If these bands are sufficiently distinct, the molecules in these zones can be examined and studied separately by fixing macromolecules and washing the electrophoretic medium to remove the buffer solution.
In order to prevent the electrophoretic medium from fracturing during the handling required for procedures commonly associated with electrophoresis it is useful to adhere the electrophoretic medium to a suitable support. A desirable electrophoretic support designed to prevent fracturing of the medium should be adherent to the medium, dimensionally stable and thermally stable.
U.S. Pat. No. 4,415,428, issued to Nochumson et al., discloses an electrophoretic support comprising a base plate having on at least one side thereof, a layer of an adherent resin. The adherent resin is derivatized to include ethylenically unsaturated groups capable of undergoing copolymerization with acrylamide. Supports coated with these adherent resins are particularly useful for bonding to a polyacrylamide gel cast on the support.
In U.S. Pat. No. 4,415,428, the adherent resins are derivatized in reactions with acylating or allylating reagents that include 2 to 12 carbons and at least one ethylenically unsaturated member. The aldehyde end groups of polysaccharide resins must be blocked to prevent the polysaccharides resins from being discolored as a result of acylation or alkylation. Following derivatization, the derivatized adherent resin must be purified and any solvents removed. However, there is still the risk that the residue left by the derivatization process may contaminate the electrophoretic buffer solution and the derivatization process may cause the resin to become discolored.
It is also known that a small percentage, typically 10% or less, of unmodified agarose lots purchased commercially are useful as adherent resins for electrophoretic supports under limited conditions. The small percentage of unmodified agarose lots useful as adherent resins are typically low electroendosmosis (LE) agarose, although a smaller percentage of middle electroendosmosis (ME) agarose lots are useful as adherent resins. Thus, commercially purchased agarose lots are commonly sampled to determine whether they posses the necessary properties to bind a polymeric support to an electrophoretic medium.
When unmodified agarose lots are used as adherent resins, an electrophoretic support is generally formed by applying an agarose solution to a corona “treated” polymeric support. Once the agarose solution dries, an electrophoretic medium may be cast directly upon the polymeric support. Approximately 10% of the unmodified agarose lots purchased commercially provide sufficient bonding to act as an adherent resin. Prior to the technique described herein, there was no way of determining, except through trial and error, whether a specific agarose lot possessed the necessary properties, except to coat the agarose onto a polymeric support, cast an electrophoretic medium on the agarose coating and evaluate the bonding. Similarly, prior to the claimed there is no known method of making a larger percentage of commercially purchased agarose lots useful as adherent resins for electrophoretic supports.
Thus, a need exists for a method of making a larger number of agarose lots useful as the adherent resin for an electrophoretic support. A need also exists for a method of malting an electrophoretic support with an adherent coating that creates a reduced risk of contamination or discoloration.